Rotor Fluorogenic D-amino acids
Rotor Fluorogenic D-amino acids (RfDAAs) are second-generation FDAAs that are designed for time-lapse applications for peptidoglycan studies. FDAAs have demonstrated powerful applicabilities in visualizing peptidoglycan formation and its synthesis activity. However, the need of washing out excess FDAAs from the solution to reduce background fluorescence noise significantly reduced the temporal resolution of the labeling experiments. The washing processes, usually conducted by centrifuging, could also disrupt cell growth and results in artificial effects. To address these issues, rotor fluorogenic D-amino acids are designed and synthesized. RfDAAs remain non-fluorescent in solution but become fluorescent upon the incorporation into peptidoglycan. Their fluorescence is triggered by the increased sterical hindrance in the peptidoglycan structures. Therefore, RfDAA labeling can be visualized without washing steps and cell fixation, making them great tools to study transpeptidase activity in time-lapse manners.
Comparison between FDAA and RfDAA labeling. [1]
RfDAA labeling does not require washing steps. [1]
Mechanism of FDAA labeling
Rotor Fluorogenic D-amino acids belong to monopeptide probes. Therefore, they share the same incorporation mechanism with FDAAs. Their incorporation is conducted by transpeptidases of the cells so RfFDAA labeling reflects peptidoglycan cross-linking activity. Inhibiting transpeptidases activity results in reduced RfDAA labeling. [1] Also, the L-enantiomer of RfDAAs do not label peptidoglycan. RfDAAs incorporate into the 5th position of peptidoglycan polypeptides.
Applications of RfDAAs
RfDAA labeling does not require cell washing and fixation steps. Their signal can be visualized by microscopy right after the probe treatment. In this case, we can minimize the disturbance of cell growth and the resulting artificial effects in the experiments. One example of short-pulse RfDAA labeling from Streptomyces venezuelae without washing and fixation is shown above. Another remarkable application of RfDAA is their use for time-lapse tracking of peptidoglycan growth. By growing bacterial cells on a microscope system, we successfully observed peptidoglycan formation step by step. This is because newly synthesized peptidoglycan structures contain high transpeptidase activity which incorporates RfDAAs into the structures and turns them on. RfDAAs are the first and the only probes that enable time-lapse monitoring of peptidoglycan formation in vivo.
Yellow arrowhead: newly formed septa; white arrowhead: newly formed cell branches.[1]
In addition to in vivo peptidoglycan labeling, RfDAAs are found to be useful for in vitro study of transpeptidase activity. Transpeptidases are valuable targets for antibiotic developments. Quantitative analysis of transpeptidase activity in vitro has been difficult because HPLC (high-performance liquid chromatography) and MS (mass spectrometry) are usually required for analyte identification in current analysis assays. These processes are time-consuming and require more human effort, impeding their high-throughput applications. RfDAAs, however, can report transpeptidase activities in vitro without the need of analyte isolation and identification. They provide fluorogenic responses when they are incorporated into synthetic peptidoglycan substrate through transpeptidase's activity. This approach simplifies the protocol of the analyses and thus significantly reduces the cost and time required for high-throughput applications.
Scheme of in vitro transpeptidase activity assay. [1]
RfDAA spectra and microscopy settings
RfDAAs have a large stoke shift compared to most commercially available fluorophores. Therefore, customization of microscope condition is usually required for visualizing RfDAA signal. Briefly, Rf420DL can be visualized using a DAPI excitation filter (400 nm) and a FITC emission filter (490 nm); Rf470DL can be visualized using a FITC excitation filter (470 nm) and a Cy3 emission filter (610 nm); Rf490DL can be visualized using a FITC excitation filter (470 nm) and a Cy5 emission filter (725 nm).
Excitation and emission spectra of RfDAAS [1]
Photochemical properties of RfDAAs [1]
References
[1] Hsu et al. Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays. Nat. Chem. 2019, 11, 335-341.